Polymerase chain reaction (PCR) was developed in the 1980s and is today the most commonly used method for amplifying DNA and has become a central tool in both synthesis and analysis of DNA in a multitude of applications.
In traditional PCR amplification, the sample or source material is pretreated in order to extract and purify DNA. It is also desirable to remove or neutralize various PCR inhibitors present in the sample or source material, as it is well known that wild-type Taq polymerase is easily inhibited by cellular debris.
Newly developed more robust polymerase enzymes have however made it possible to amplify DNA directly from a crude or minimally treated sample or source material. This approach has been coined “direct PCR”. Direct PCR has great potential for use in various diagnostic applications, where a simplified sample handling and faster turnaround times from sampling to results would give significant benefits to both the user and the patients.
While potentially offering savings in time and cost, quantitative direct PCR also has some problems to overcome before it can be used in diagnostic and environmental applications, where the typical samples such as saliva, buccal swabs, tissue samples, blood, cell culture, plant material, soil samples etc often contain a complex mixture of organic and inorganic debris.
The general procedure for performing direct PCR on complex samples usually involves an initial denaturation to release DNA for amplification. It is suggested that the results can be improved by performing the denaturation in a suitable buffer, spinning down the cellular debris and using the cleared supernatant in the PCR. This however requires an additional step of collecting the supernatant and transferring it to a system for performing the PCR.
When direct PCR is performed in a heating block, the central element of all conventional PCR instruments, real time quantitative PCR (qPCR) requires that excitation light is directed into the sample container, and emitted light read from the same direction. Frequently this is done from above, as only the opening of the container is available, when the remaining container is surrounded by the heating block. In a crude sample, the fluorescence is attenuated and scattered by particulate matter, such as cell debris, present in the sample. Further, reflected excitation light may interfere with the reading of the emission light, and filter arrangements may be necessary to eliminate reflected excitation light.
FIG. 1, panel A, schematically illustrates the above described background art, showing a PCR tube 101 containing a sample 102 placed in a heating block 103. A light source 104 and a detector 105 are positioned above the opening of the PCR tube.
There are only a few automated, rotary systems, such as the Rotor Gene® (formerly Corbett Life Sciences Inc., now Qiagen Inc.) or the QuanTyper™-48 instrument (AlphaHelix Molecular Diagnostics AB), capable of performing qPCR. In these, excitation light is emitted by a light source positioned substantially at a right angle to the sample container, and emission light is detected by a sensor positioned at the apices of the container, detecting light emitted along the longitudinal axis of the containers. The rounded bottom or apice of the sample containers, frequently commercially available PCR-tubes, is used as a lens, collecting the emission light.
FIG. 1, panel B, schematically illustrates the above described background art, showing a PCR tube 101 containing a sample 102 placed in a rotor 110. A light source 104 and a detector 105 are arranged outside the rotational path of the PCR-tubes.
When qPCR is performed on a crude sample, the complex nature of the sample influences the sensitivity, reliability and repeatability of the result, in particular because of difficulties in the excitation, reading and quantification of the fluorescence.
WO 00/58013 discloses a device for performing PCR, where a significant centrifugal force is utilized to homogenize the sample with respect to temperature. The efficient homogenization makes it possible to heat the samples more aggressively, without risking over-shooting, and the uninterrupted centrifugation makes it possible to rapidly cool the samples. The result is significantly shortened ramping-times, thus minimizing the time required for performing PCR. The disclosure mentions the possibility of using a radiation source and a reflectance sensor for detecting chemical reactions in the samples, e.g. the light reflectance or emission indicating the end point of a reaction or a positive or negative test answer. The disclosure however does not address the issues of direct PCR.
GB 1 402 225 concerns a rotary dynamic multi-station photometer-fluorometer. It is suggested that the most obvious arrangement is that where a light source is disposed on one side of a sample-holding cuvette and a photodetector on the other, but that such arrangement requires appropriate filters in order to eliminate the excitation light passing through the sample. The elimination of interfering excitation light may also be accomplished by angled excitation emission detection wherein the excitation beam is oriented 90 degrees with respect to the photodetector which measures the emitted fluorescence.
GB 1 402 225 also mention another problem associated with determining solute concentration by fluorescence measurement, which occurs where the sample is characterized by relatively high absorbance. In practice, the sample itself attenuates the excitation, and this phenomenon is often referred to as the “inner filter effect”.
When performing quantitative direct PCR, handling the complex sample poses problems that existing methods and devices are not well equipped to handle. There is a need of improvements in the methods and devices for performing quantitative direct PCR in order to achieve repeatable and reliable results, in particular when performing qPCR on crude samples such as, but not limited to, saliva, buccal swabs, tissue samples, blood, cell culture, plant material, soil samples etc, which often contain a complex mixture of organic and inorganic debris. For diagnostic purposes, there is a need for improvements in the handling and analysis of in particular whole blood samples.